Although previous studies from this group have indicated the absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for virus replication, this enzymatic function has received little attention as an antiviral target. However, the finding that HIV-1 RNase H and integrase (IN) are derived from the same superfamily of nucleotidyl transferases has allowed the implementation of screening strategies based on IN inhibitors previously identified at the NCI. Several natural products have initially been identified that act on both RNase H and IN, while a limited number show selectivity for the individual proteins. Subsequently, two fluorescence-based assays have been implemented in collaboration with the NCI Molecular Targets Development Program for high-throughput screening of several NCI libraries (n = 250,000) for inhibitors of HIV-1 RNase H function (Parniak et al., 2003; Chan et al., 2004). Several classes of inhibitors, active at submicromolar concentrations, have been identified with enhanced specificity for HIV-1 RNase H. The hydroxylated tropolone beta-thujaplicinol was identified as a potent HIV-1 RNase H inhibitor that most likely functions via disturbing metal ion coordination at the active site (Budihas et al., 2005). More importantly, beta-thujaplicinol was demonstrated to synergize with a nonnucleoside-based RT inhibitor, which has important clinical implications. Presently, the binding site for inhibitors of the HIV-1 E.coli and HIV-1 RNases H is being determined by a combination of X-ray crystallography and NMR spectroscopy. A patent on a selected group of compounds with antiviral activity has also been submitted.